Iron is an essential functional component of heme and hemoglobin and is required for red blood cell synthesis. During increased red cell production, intestinal iron absorption and the release of iron from stores are increased, facilitating the production of new erythrocytes. The liver produced hormone hepcidin controls the major flows of iron into plasma: absorption of dietary iron in intestine, recycling of iron by macrophages, which ingest old erythrocytes and other cells, and mobilization of stored iron from hepatocytes. Following erythropoietic stimulation, hepcidin rapidly decreases causing more iron to be delivered for red blood cell synthesis, but the mechanism was unknown until recently. We identified the erythroid hormone erythroferrone (ERFE), an orphan member of the C1q-TNF family of proteins, and presented evidence that it regulates hepcidin during intensified red blood cell production. ERFE is a potent (EC50~100 pM) suppressor of hepcidin, is produced by erythroid precursors in response to erythropoietin (EPO) and is required for rapid suppression of hepcidin after hemorrhage or EPO administration. The targets of ERFE are the hepcidin-secreting hepatocytes. The molecular mechanism by which ERFE suppresses hepatic hepcidin expression is unknown and is the subject of this proposal. The specific aims of the proposed project are: Aim 1) To characterize the structure-function relationship of ERFE. We will generate and test truncated forms of ERFE and collagen domain ERFE mutants to determine the structural features that are important for ERFE function in vivo and in vitro. We will also determine ERFE's oligomerization state in circulation by blue native PAGE. We expect the TNF-like domain of ERFE to be important for its activity and the hepcidin-suppressive form of circulating ERFE to be a trimer. Aim 2) To identify the ERFE receptor. We will use progressively more general strategies starting from a candidate receptor screen to unbiased proteomic analysis of ligand-receptor complexes. Based on ERFE similarity to C1q-TNF family of ligands whose known receptors are either progestin and adipoQ receptors (PAQRs) or members of the TNF receptor family, we will first complete our so far negative PAQR screen. We will then use an ELISA-like assay to screen a TNF receptor family library using ERFE as the test ligand. If these approaches do not yield verified receptors, we will proceed to screening expression libraries with ERFE and to proteomic analysis of ERFE complexed with its biological receptor in a hepatocyte cell line. Aim 3) To determine the signaling pathway by which ERFE suppresses hepcidin. We will use phosphopeptide mass spectrometry and promoter mutational analysis to link the putative signaling pathway with its associated transcription factors that bind to the hepcidin promoter. We expect ERFE to signal through pathways similar to those of other TNF family members, such as the MEK/ERK pathway. Targeting the hepcidin-suppressing erythroid hormone pathway may offer improved treatments for patients with hepcidin- related iron disorders.